Body: We report the case of a 42-year-old female patient diagnosed with intermediate-risk AML in April 2024. After first remission, she underwent a 9/10 HLA-mismatched unrelated allogeneic transplant (mismatches in HLA-DRB1 and -DPB1) in August 2024. No donor-specific antibodies were detected, although multiple antibodies were present in the panel reactive antibody screen. In April 2025, disease relapse was confirmed with 30% blasts in bone marrow. Immunophenotyping showed expression of CD7, CD13, CD33, CD34, CD117, HLA-DR, and cyMPO. STR-PCR chimerism revealed only 33% donor origin. Given the plan for a second transplant following rescue, we investigated potential HLA loss, considering the impact this mechanism can have on donor selection and treatment decisions. In our lab’s routine, magnetic bead-based CD34+ cell selection is the standard approach for isolating leukemic cells from blood or bone marrow specimens. However, due to weak CD34 expression in this case, we also performed cell sorting (CytoFLEX SRT, Beckman Coulter) using CD117+ (a marker with moderate expression in the blasts) as an alternative method to enrich the cell population of interest. DNA extracted from CD34+ bead isolation yielded insufficient DNA concentration for further testing. In contrast, cell sorting based on CD117+ provided both adequate DNA concentration and high cell purity, allowing us to proceed with intermediate-resolution HLA typing, which ruled out HLA loss as the mechanism of relapse. Additionally, STR-based chimerism analysis on the sorted CD117+ cells showed 0% donor origin, confirming the blast cell purity.
Conclusion: We report a case in which cell sorting played a critical role in investigating HLA loss-mediated relapse following mismatched unrelated donor transplantation. Routine methods like CD34+ magnetic bead isolation may be limited by low leukemic cell yield and poor antigen expression - challenges evident in this case. By using CD117+ cell sorting, we overcame these limitations and obtained high-quality material for this essential assessment, without the need for algorithm-based analysis. As HLA loss has important clinical implications for prognosis and therapeutic planning, employing precise cell isolation strategies is essential. This approach enabled our investigation and directly impacted clinical decision-making in a complex transplant scenario.
